Interaction of Xenorhabdus nematophilus subsp. nematophilus with the haemolymph of Galleria mellonella

The lethal dose (LD)₅₀ values and probit-mortality regression slopes of the primary and secondary forms of Xenorhabdus nematophilus subsp. nematophilus for Galleria mellonella were equal. The two bacterial forms grew at equal rates in larval serum-supplemented media. The secondary form grew less well in larval serum-supplemented media than in synthetic larval serum.The secondary bacteria adhered to the haemocytes to a greater extent than did the primary bacteria. Both types of bacteria did not produce metabolites suppressing the ability of the haemocytes to respond to Bacillus cerues.Differences were observed in the rate of clearance of the primary and secondary bacteria from and their subsequent re-entrance into the haemolymph in vivo. This appeared to be independent of bacterial metabolism. Evidence is presented showing multiplication of the primary bacteria during their association with the haemocytes.The total haemocyte counts increased during bacterial infection. All the haemocytes were killed. The rate and pattern of change of the total haemocyte counts was influenced by the form of bacteria and independent of bacterial metabolism.     

Identification, cloning and expression of a second gene (vpr1) from the venom of the endoparasitic wasp, Pimpla hypochondriaca that displays immunosuppressive activity

Previously, we biochemically isolated an immunosuppressive protein (VPr3) from the venom of Pimpla hypochondriaca and cloned and expressed the gene in bacteria. The deduced amino acid sequence for VPr3 shares 63% identity with a second P. hypochondriaca protein, venom protein one (VPr1). We have now cloned and expressed the gene for vpr1. The expression of His-tagged recombinant VPr1 (rVPr1) in E. coli BL21 Star™ (DE3) cells was induced by the addition of 0.5 mM IPTG. Cultures were grown at 24 and 37 °C, and VPr1 more readily partitioned into the soluble fraction at 24 °C. Soluble rVPr1 was purified using the MagneHis purification system and a modified elution buffer to allow the protein to be directly tested for activity against haemocytes. It was observed that rVPr1 prevented the ability of haemocytes to spread and form aggregates in vitro in a dose-dependent manner. Furthermore, comparable levels of activity were observed when similar concentrations of rVPr1 and rVPr3 were tested. In addition, the encapsulation of Sephadex beads in vivo was reduced by the presence of rVPr1 and beads were unencapsulated (negative) or only weakly encapsulated. The functional and physio-chemical properties of rVPr1 and rVPr3 are compared and discussed.     

Effects of infection of EGFP-expressing Escherichia coli on haemocytes in Ciona intestinalis

The effects of infection of EGFP-expressing Escherichia coli on the haemocytes of the ascidian Ciona intestinalis were investigated. The results showed that THC of the infected individuals changed significantly. Hyaline amoebocytes phagocytosed E. coli in 5 min and excreted lysosome particles that attached to the surface of the bacteria. Granular amoebocytes released lots of particles for humoral immunity while stem-cell-like haemocytes remained intact. With the increase in THC, the stem-cell-like haemocytes showed division and proliferation. A small portion of hyaline amoebocytes was at early apoptosis stage 1 h after infection and typical apoptosis bodies emerged in granular amoebocytes. A few of the infected haemocytes showed DNA damage using SCGE assay. Flow cytometry analysis revealed an obvious apoptosis peak in infected haemocytes. In conclusion, apoptosis was found to be an important immune response of ascidian haemocytes response to bacterial infection. To our best knowledge, this is the first report of the occurrence of apoptosis of haemocytes in ascidians.     

The encapsulation of foreign tissue implants in Galleria mellonella larvae.

When implanted into the haemocoel of the wax moth, Galleria mellonella, fragments of nerve cord from the locust Schistocerca gregaria were shown to be encapsulated by host haemocytes, in a two phase system. The initial reaction to the implant was observed within 5 min of implantation and involved the contact and lysis of the granular haemocytes on the nerve cord, and the release of granular material around it which resulted in localised clot formation.The second phase involved plasmatocyte adhesion to the implant ca. 30 min after its introduction into the haemocoel, but only at sites of granular cell lysis. These cells continued to be added to the capsule until 60 to 72 hr after implantation, and during this time granular release continued, although no further granular cell lysis occurred. The complete capsule (72 hr) could be divided into three distinct regions of cells showing various modifications from free plasmatocytes.It is suggested that encapsulation in this species involves some chemotactic element which originates from the granular cells and acts specifically on the plasmatocytes.     

Differential adhesion of the haemocytes of Leucophaea maderae (Blattaria) to a glass surface

A system for the study of insect haemocytes in vitro is described. The system was used to analyse the adhesive properties of the haemocytes of the cockroach, Leucophaea maderae. The two main types of haemocytes, plasmatocytes and granulocytes, showed considerable differences in adhesive properties, which allowed the production of nearly homogeneous monolayers consisting of either plasmatocytes or granulocytes. The much stronger adhesion of the plasmatocytes is discussed in relation to their role in phagocytosis and encapsulation.     

Himasthla elongata: Effect of infection on expression of the LUSTR-like receptor mRNA in common periwinkle haemocytes

The first mollusc mRNA coding G-protein-coupled transmembrane receptor (GPСR), homologous to human receptors LUSTR 1 (GPR107) and LUSTR 2 (GPR108), was isolated from haemocytes of common periwinkle Littorina littorea. The analyses showed that the full-length cDNA is 1935 bp long and is predicted to encode a 614 amino acid protein (named Lit-LUSTR) with a calculated molecular mass of 69.6 kDa and theoretical isoelectric point 7.59. Pair-wise comparisons between Lit-LUSTR and LUSTR proteins from human or mouse have approximately 38% identity and 56% similarity. Lit-LUSTR clusters with LUSTR-A sub-family proteins and is a first characterization of proteins containing Lung7TM-R domain in Mollusca. Significant differences were found between the Lit-LUSTR mRNA levels in haemocytes of healthy periwinkles and those naturally infected with the echinostome trematode Himasthla elongata. Down regulated expression of the LUSTR-like receptor caused by infection illustrates modification of the haemocyte receptor system and may be attributed to the previously demonstrated greater numbers of “immature” haemocytes in the circulation of infected snails.     

Exposure to anoxia of the clam, Chamelea gallina: II: Modulation of superoxide dismutase activity and expression in haemocytes

In order to investigate the influence of anoxic stress on haemocyte immune response, specimens of Chamelea gallina were exposed to 24 and 48 h anoxia. To evaluate recovery capacity, clams were maintained, at the end of the anoxic phase, for 24 h in reoxygenated seawater. In this paper, activity and expression of the antioxidant enzyme superoxide dismutase (SOD) were studied on haemocyte lysate and haemolymph. Reported results have shown that the anoxic stress changed strongly the response of C. gallina blood cells. Indeed, at the end of the anoxic phase in both experiments (24 and 48 h of anoxia exposure), SOD activity in haemocyte lysate decreased significantly with respect to the control, likely because of a decreasing superoxide anion generation in anoxia. Expression analyses were coherent with activity values.In the first experiment (24 h anoxia), reoxygenation determined an increase in activity of both Cu/Zn-SOD and Mn-SOD, but with values that remained significantly lower than those of the controls. It seems that after the applied anoxic stress, 24 h of recovery is not sufficient to restore pre-anoxic conditions. In the second experiment (48 h anoxia), SOD isoforms showed a different response during the recovery of animals. Cu/Zn-SOD activity dropped below the values showed by haemocytes of anoxic bivalves, while Mn-SOD activity values exceeded significantly those of controls. The different haemocyte response could be probably due to a further stress suffered by the clams because of a massive spawning during the reoxygenation phase. Therefore, the high values of activity shown by Mn-SOD during the recovery are likely to be due to the high inducibility of this isoform.In Cu/Zn-SOD expression analyses, two immunoreactive bands were highlighted in both experiments. The former (apparent molecular weight of 16 kDa) corresponds to the expression of SOD1 and the latter (apparent molecular weight of 28-30 kDa) could be attributed to EC-SOD (SOD3), a Cu/Zn-SOD isoform located in extracellular ambient and identified both in vertebrates and invertebrates. The strong SOD3 expression during anoxia exposure and the further spawning stress (second experiment) testified its inducibility in C. gallina haemocytes and haemolymph in response to stressful conditions.     

Studies on prophenoloxidase and protease activity of Blaberus craniifer haemocytes

Using a citrate-EDTA buffer as an anticoagulant it was possible to isolate intact haemocytes from the insect, Blaberus craniifer, without causing extensive degranulation and subsequent clotting. A haemocyte lysate from this insect contained prophenoloxidase (proPO), which could be activated by β 1,3-glucans. The activation process was dependent upon Ca²⁺ ions and seemed to occur by a limited proteolysis, since several serine protease inhibitors such as soybean trypsin inhibitor, benzamidine and $\text{p-}\text{nitrophenyl}\text{-p′-}\text{guanidobenzoate}$ blocked convertion of proPO to the active enzyme. Treatment of proPO with urea or heat also caused proPO activation but probably without the intervention of serine proteases, since the protease inhibitors used failed to block the activation. Within the haemocyte lysate, several endopeptidases were present, which were enhanced in activity by prior treatment with β 1,3-glucans. These endopeptidases were inhibited in activity when the haemocyte lysate was incubated with benzamidine prior to the addition of β 1,3-glucan. This provides further indications that the activation of proPO involves a limited proteolytic attack. The active phenoloxidase enzyme became strongly bound to foreign surfaces and this phenomenon may assist in providing opsonic properties for the proPO cascade.     

Iron contributes to the antibacterial functions of the haemolymph of Galleria mellonella

Studies with Galleria mellonella larvae and the iron chelating agent EDDA showed that iron was essential for the removal of dead Xenorhabdas nematophila and Bacillus subtilis from the haemolymph. The delay in removal of the bacteria from the iron-restricted haemolymph was attributed to reduced adhesiveness of the haemocytes and prophenoloxidase activity. Iron augmentation returned these activities to control levels. Whereas dead B. subtilis had no effect on the concentration of ferrozine-detectable iron (henceforth iron) in the haemolymph, dead X. nematophila was associated with substantially lower levels of iron as the number of damaged haemocytes increased. Haemocyte lysate lowered the concentrations of iron in both FeCl₃ solutions and deproteinized larval serum independent of serum lipids. Haemocyte lysate added to tryptic soybroth lowered the level of iron and limited the growth of X. nematophila. X. nematophila limited iron availability in the plasma by releasing lipopolysaccharides; such a mechanism may be a means of impairing the antimicrobial defences of the insects.     

Growth-promoting effect of haemocytes on insect epidermis in vitro

The epidermis of 21-day-old leg regenerates of cockroaches (Leucophaea maderae) was cultivated in vitro. Outgrowth of the epidermis only occurred in connexion with haemocytes.Haemocytes contaminating the epidermal explants show strong adherence to epidermal cells. The epidermal cells adhering to moving haemocytes are stretched out to long projections or completely pulled out of the epithelium. When more haemocytes are present, they can form an uninterrupted line at the margin of the epidermis. By the adhesion of marginal cells of the epidermis to the moving haemocytes, the epithelium is apparently pulled out into broad tongues. In these tongues the epidermal cells become highly flattened, especially at the front, and soon begin to divide. Outgrowth in the tongues continues only as long as there are haemocytes at the front. When they have disappeared, outgrowth stops, the flattened epidermal cells detach from the glass surface, round up, and the outgrown tissue may withdraw again.For further analysis of the interactions of haemocytes and epidermal cells the epidermis is placed on a monolayer of haemocytes. The epidermis rapidly grows out on such a monolayer. The epidermal cells either move over or under the haemocytes indicating that there are substances on both sides of the haemocytes which are attractive to the epidermal cells and cause their flattening and outgrowth. Similar outgrowth occurs on fixed monolayers of haemocytes. There is no outgrowth on areas where the monolayer has been scraped away. No principal differences can be found between monolayers consisting almost exclusively of either plasmatocytes or granular haemocytes.The similarities of the observed interactions of haemocytes and epidermal cells to encapsulation and wound healing are pointed out. A hypothesis is presented which assumes that the haemocytes during wound healing not only serve as a mechanical support but also as a chemical guide by which the closure of the wound by epidermal cells is enhanced.     

Phagocytosis of inert particles in Locusta migratoria and Galleria mellonella: Study of ultrastructure and clearance

Inert particles (iron saccharate or latex beads) injected in the haemocoel of Locusta migratoria, are taken up by pericardial cells (iron saccharate only), reticular cells of the haemopoietic tissue and certain haemocytes: plasmatocytes and coagulocytes; these two haemocyte types are also the main phagocytic blood cells in Galleria mellonella.Necrosis of phagocytic haemocytes, following injection of an overdose of iron saccharate, explains the profound modifications of the haemogram observed during the first 24 hr following injection; the macrophagic evolution of reticular cells slows down the haemopoietic differentiation of these cells and explains the long term disturbances of the blood picture.Clearance of latex beads injected in larvae of Locusta complies to an exponential function of time; we can determine a granulopectic index which will permit comparisons to be made between clearance of inert and of ‘antigenic-like’ particles.     

Prostaglandins inhibit testosterone secretion by mouse testes in vitro

Production of testosterone (T) by decapsulated mouse testes in vitro was significantly inhibited by adding prostaglanain (PG) A₁, PGA₂ or PGE₁ to the incubation medium. Prostaglandin A₁ at a concentration of 10^?6M inhibited T production in this system both in the presence of moderate amounts of hCG (12.5 or 25.0 mIU/ml), and in the absence of gonadotropins. However, in the presence of very high levels of hCG (125.0 mIU/ml), all PGs tested appeared to have had a slight potentiating effect on T production when added in concentrations ranging from 10^?7 to 10^?5M, and the inhibition of T accumulation in the medium was consistently observed only when the concentration of PGs was increased to 10^?3M. These results suggest that a direct effect of PGs on testicular steroidogenesis may account for, or contributes to, the decrease in peripheral T levels observed after administration of PGs in vivo.     

L'encapsulation hémocytaire expérimentale chez le lépisme Thermobia domestica

The introduction of inert foreign objects into the thorax of the thysanuran Thermobia domestica provoked the formation of a cellular capsule, the development and fine structure of which were examined.Encapsulation at first simply results from the accumulation of blood cells around the implant. It is possible to distinguish 48 hr later four regions in the cellular capsule: (1) An exterior layer including normal haemocytes. (2) An intermediate layer formed by homogeneous intercellular electron-dense material and by stretched haemocytes. These haemocytes have numerous microtubules, without any granular particles, and are linked together by desmosomes. (3) An interior layer of cells in the process of necrosis and rich in lysosomes. (4) A very thin limiting layer tentatively interpreted as melanin.The large number of haemocytes devoid of the specific features of the fibroblasts and the very important reduction of the acellular material without collagen fibrils distinguish clearly the cellular capsules of the Insecta from the granuloma of the Vertebrata and other groups.     

Brain hormone carrier haemocytes in the moth Monema flavescens

The movement of neurosecretory substances released from the neurosecretory B cell in the pars intercerebralis to the haemolymph was examined with the progress of the termination of diapause in the slug moth pharate pupa, Monema flavescens.The injection of precipitates in the haemolymph of the pharate pupa just before the termination of diapause into diapausing pharate pupae reduced the numbers of days required for them to pupate. In the precipitates, seven types of haemocytes were present. The number of haemocytes, especially the granular cell, increased just before the termination of diapause. AF and CHP positive substances not detected in the haemocytes of diapausing pharate pupae appeared in the granular cells just before the termination of diapause. The period also coincided well with the releasing period of the neurosecretory B cell. Histological examination showed that granular haemocytes gathered around the pars intercerebralis at this period and exchange of neurosecretory substances occurred between granular haemocytes and neurosecretory B cells. Then granular haemocytes migrated to the region of the prothoracic gland. From digestion tests of the neurosecretory substances with rabbit serum and from the implantation tests of the neuroendocrine system, the substances detected in both the neurosecretory B cell and the granular haemocytes seemed to be the same. The dye injection caused a delay in larval-pupal ecdysis emergence. Droplets of black ink are incorporated into the granular haemocytes. This seems to be caused by blocking of the transport of neurosecretory substances released from cytoplasmic processes of the neurosecretory B cell.From these experiments, it is suggested that neurosecretory substances of the prothoracotropic hormone are transported to the prothoracic gland, along with granular haemocytes, after being released directly from the neurosecretory B cell to the haemolymph.     

The specificity of the serum agglutinins of Periplaneta americana and Schistocerca gregaria and its relationship to the insects' immune response

The agglutinating activity of insect serum against vertebrate erythrocytes has been examined for two insect species, the cockroach Periplaneta americana and the locust Schistocerca gregaria. Differences were found between the two insect species, in that cockroach serum agglutinated a wider range of erythrocyte types than did locust serum and the titre of the agglutinating activity of cockroach serum was higher in all cases. The results of attempts to inhibit the agglutinating activity using a variety of sugars and glycoproteins revealed that the combining specificities of the agglutinating molecules of the two species differed. Agglutination of rat erythrocytes by cockroach serum was not inhibited by any of the sugars or glycoproteins tested, whereas several of these compounds, in particular sucrose, partially inhibited the agglutination of rat erythrocytes by locust serum.The significance of these results is discussed in relation to the observation that haemocytes of the cockroach respond to a wider range of transplanted tissues in vivo than do those of the locust.     

Effects of the parasitization of a braconid, Apanteles, on the blood of its host, Pieris

Parasitization of a braconid wasp, Apanteles glomeratus, of larvae of a common cabbage butterfly, Pieris rapae crucivora, caused changes in differential haemocyte count (DHC), total haemocyte count (THC), and encapsulative capacity against dead eggs of Apanteles in the fourth and fifth instar host larvae.However, no correlation could be found between the number of Apanteles eggs deposited and THC of the middle fourth instar host larvae or between the number of parasitoid larvae and specific gravity of the haemolymph from the late fifth instar host larvae.From the changes in DHC and in THC of both non-parasitized and parasitized Pieris larvae, an increase in the number of plasmatocytes of non-parasitized Pieris larvae in the early fourth instar period was supposed to be due to transformation of prohaemocytes into plasmatocytes, and a low population of plasmatocytes of parasitized larvae in the comparable period was assumed to be due to a suppression of transformation of prohaemocytes by some factor released from the parasitoid eggs.Failure of the parasitized fourth instar Pieris larvae to encapsulate injected dead eggs of Apanteles indicated that the parasitoid embryos were, in some way, actively inhibiting the encapsulation reactions of the host.The increase in THC of the parasitized fifth instar larvae could not be ascribed to a decrease in the volume of host haemolymph. Rather it could be interpreted by a suppression of adhesive capacity of haemocytes in the host haemocoel to tissue surfaces.Reduced encapsulative capacity of the parasitized fifth instar larvae might be attributed either to a depression of the adhesive activity of plasmatocytes resulting from a depletion of energy source for haemocytes in the host haemolymph by parasitization, or from an active suppression of adhesiveness of the plasmatocytes by secretions from ‘giant cells’ (teratocytes) originated from the parasitoid.     

Entamoeba histolytica: Molecular cloning and characterization of a novel neutral sphingomyelinase

A novel neutral sphingomyelinase (nSMase) was characterized in Entamoeba histolytica trophozoites. SMase, a sphingomyelin-specific form of phospholipase C, catalyzes the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Three amebic putative nSMase genes were found to be actively transcribed. Mg²⁺-independent nSMase activity in the soluble fraction of the trophozoites was stimulated by Mn²⁺ and partially inhibited by Zn²⁺. nSMase activity of the recombinant protein EhnSM1, increased 4.5-fold in the presence of 0.5 mM Mn²⁺, and abolished by 5 mM Zn²⁺. A dose-dependent inhibition of rEhnSM1 was observed with scyphostatin, a specific inhibitor of nSMases. The EhnSM1 and EhnSM3 were detected in the soluble fraction of the amebic lysate as 35-37 kDa proteins by western blot analysis. Immunofluorescence assay showed that the overexpressed HA-tagged EhnSM1 and EhnSM3 were localized to the cytosol. The biological role of these novel E. histolytica nSMases described in this work remains to be determined.     

Haemocytic encapsulation and the prophenoloxidase-activation pathway in the locust Schistocerca gregaria forsk

Negatively-charged Sepharose beads are not encapsulated in vivo by haemocytes of the locust Schistocerca gregaria. It has been suggested by other workers that components of the prophenoloxidase activation pathway of haemolymph might adhere to foreign surfaces and stimulate haemocyte adhesion, so one possible reason for the lack of encapsulation of beads might be due to failure of these components to adhere to the bead. Beads were thus incubated in locust haemocyte lysate supernatant, in which the prophenoloxidase pathway had been activated by Ca²⁺ or Zymosan supernatant, and were then injected into the haemocoeles of locusts. Although at least 5 proteins, including phenoloxidase, could be shown to be attached to the beads, these coated beads were not encapsulated suggesting either that the putative opsonin did not attach or that none of the components is opsonic in this system.In addition, it has been shown that the prophenoloxidase pathway in locust haemocyte lysate supernatant can be partially activated in the presence of Ca²⁺, strongly activated by β1,3-glucans and that production of phenoloxidase is not enhanced by the presence of bacterial LPS and is inhibited by a serine protease inhibitor. The changes in protein composition of unactivated and activated haemocyte lysate supernatant are discussed.